Vitamin A has been shown to be an essential extracellular regulator for the normal development of germ cells in the mammalian testis. It is the long term objective of this proposal to Investigate the molecular mechanism by which retinol, a form of Vitamin A, regulates the Process of spermatogenesis. This will be accomplished by identification of target genes for the retinol- modulation in the testis. When rats are fed on a Vitamin A-deficient diet, their testes atrophy and their seminiferous epithelium degenerate. This can be completely reversed by an intraperitoneal injection of retinol (Morales and Griswold, Endocrinology, 12: 432.,1987). The retinol-induced testes reinitiate the spermatogenic process in a synchronized manner all along the seminiferous tubules. It is Presumed that sPecific gene Products and mRNAs are synthesized in direct response to retinol in either the Sertoli cells or in the germinal cells. In either case, the retinol-responsive mRNA transcripts or newly translated proteins are absolutely essential in the reinitiation process of spermatogenesis in the VAD rat testes. In order to investigate the functional significance of the retinol- induced mRNAs in the testes at the molecular level, they will be isolated from a cDNA library. Specifically, this will be accomplished by the construction of a Lambda cDNA library enriched in the unhybridized but retinol-stimulated sequences. The unhybridized sequences are single stranded cDNAs generated in the subtractive hybridization of the mRNAs from the retinol-induced testis with an excess of mRNAs from the VAD rat testes. These cloned cDNAs are essential to investigate the molecular mechanism of retinol regulation during spermatogenesis. They will be sequenced and characterized to identify their function in the testis. The expression of these cloned cDNAs will be investigated in the different testis cell types and during the different stages of the spermatogenic cycle. In addition, the level at which these sequences are regulated by retinol will be investigated, i.e., transcription, post-transcription or translation. To elucidate the molecular mechanism of retinol on testicular target genes, the regulatory elements for the retinol induction and testis tissue- specificity will be investigated in the genes encoding the retinol- specific proteins.